Over the past several years, we have been studying two phenomena in cloned populations of CD4+ T lymphocytes referred to as costimulation and anergy. Both affect the production of the T cell growth factor interleukin-2 (IL-2) produced by these cells. Costimulation entails a 30 to 100-fold enhancement of IL-2 production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Anergy is an anti-proliferative state that the T cell enters when it only receives a signal through the antigen- specific receptor. In this case, subsequent stimulation of IL-2 production is inhibited 20-50-fold. Our goals are to try and understand the molecular mechanisms behind these two phenomena and to explore their relevance in vivo. During the past year we have made progress in four areas: 1. We discovered that the drug rapamycin, which inhibits certain signals transmitted through the IL-2 receptor, cannot only inhibit T cell proliferation but lead to a state of anergy, even in the presence of costimulation. This state resembles the anergy induced by T cell receptor occupancy in the absence of costimulation except that the inhibition of the production of other cytokines, such as IFN-g and IL-3, is more profound. 2. We have previously identified the ?180 region of the IL-2 enhancer as critical for an anergy effect on IL-2 production. We have now shown that three different transcription factor complexes from nuclear extracts of EL4 cells (a T cell tumor) can bind to this region: ATF2/cJun; CREB-1/CREM; and cJun/OCT. A fourth band detected on electrophoretic mobility shift assays was not identified. When we examined nuclear extracts from normal T cell clones, the dominant binding complex was CREB/CREM. This complex showed increased levels of binding after T cell receptor stimulation and most interestingly showed increased binding with extracts from anergic T cell clones even in their resting state. 3. We and others have previously shown that part of the effect of CD28 signaling that enhances IL-2 production is a stabilization of IL-2 mRNA. We have now shown using sequenced-tagged constructs containing deletions, that the 3? untranslated region of the mRNA is required for this effect. The CD28-induced stability is lost when the 3? UTR is deleted. It is also abolished if a nonsense codon is introduced into the first exon of the reporter. Finally removal of the 3? UTR revealed a small CD28-destabilizing effect on the mRNA which was controlled by sequences mapping between the middle of the third exon of the gene and the stop codon. 4. We previously set up an in vivo tolerance model in which a T cell receptor transgenic mouse on a RAG2 knock-out background (to eliminate endogenous T cell receptors) was injected three times with the superantigen staphylococcal enterotoxin A (SEA). We now have identified two separate mechanisms that are responsible for the inhibition of IL-2 production and T cell proliferation following stimulation of the spleen cells with antigen and presenting cells in vitro. One is a cytokine-induced suppressive mechanism mediated by CD4+ cells producing TGFb and IL-10. The other is an anergic state in which the amount of IL-2 production per cell is decreased 60% and the response to IL-2 is inhibited. The first mechanism peaks 4 to 5 days after the last in vivo immunization with SEA and then diminishes to very low levels by day 20, at which time the second mechanism is still fully present.